Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.

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We identified and characterized two chitinases, named MpCHIT1 and MpCHIT2, from the fungus Moniliophthora perniciosa - the etiologic agent of witches' broom disease in cacao tree (Theobroma cacao L.) - during its development, mainly in the mycelia phases preceding the basidioma formation. The expression of MpCHIT1 and MpCHIT2, together with MpCHS and MpATG8 (chitin synthase and autophagy genes, respectively), was analyzed during the M. perniciosa growth and development on bran-based solid medium as well as in liquid medium containing H2O2 or rapamycin (oxidative and nutritional related-autophagy stress agents, respectively). In order to link the expression of chitin metabolism-related genes to nutritional composition influencing fungus development, we also quantified total and reduced sugars, as well as macro- and micronutrients in the bran-based solid medium. The expression analysis showed that the MpCHS expression increased through mycelial development and then decreased in the primordium and basidioma phases, while the expression of MpCHIT1 and MpCHIT2 was higher in basidioma and primordium phases, respectively. Moreover, the expression pattern of MpCHIT1 and MpCHIT2 is distinct, the second correlated with the MpATG8 expression pattern and possibly with autophagy process, while the first may be related to the basidioma formation. The quantification of total and reduced sugars, as well as macro- and micronutrients supported the idea that the cell wall restructuration due to MpCHS, MpCHIT1 and MpCHIT2 is related to stress and fungal nutrient reallocation, allowing the formation and development of the basidioma. Experiments involving M. perniciosa growth on liquid medium containing H2O2 or rapamycin showed that MpCHIT1 and MpCHIT2 were over-expressed in response to oxidative but also to nutritional related-autophagy stresses. Interestingly, the expression level of MpCHS, MpCHIT1 and MpCHIT2 in presence of rapamycin is similar to the one observed in the primordium and basidioma from bran-based solid medium. The analysis of the overall data allowed designing a general scheme of chitin metabolism and autophagy during M. perniciosa development, focusing on the mycelium phases as crucial and environmentally influenced steps preceding the primordium and basidioma formation. These data support the idea that the nutritional environment of M. perniciosa influences its development and life cycle.

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ABSTRACT. Moniliophthora perniciosa (Stahel) Aime and PhillipsMora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene 1945 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (2): 1944-1950 (2013 M. perniciosa mitochondrial plasmid and oxidative stress expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack.